Background : Sepsis is characterized by a dysregulated host response. Patients with prior solid organ transplantation (SOT) are highly susceptible to infection due to chronic immunosuppression, yet exhibit improved survival once sepsis occurs. The underlying mechanisms remain poorly understood. Methods : We analyzed a prospective multicenter cohort of septic intensive care unit patients (SepsisDataNet.NRW; n=538). Clinical outcomes were assessed using Kaplan-Meier analysis and multivariable Cox regression. Immune profiling included serum cytokines, flow cytometric immunophenotyping, plasma proteomics, and intracellular signaling using proximity ligation assays (PLA) in peripheral blood mononuclear cells (PBMCs). To reduce clinical confounding, ex vivo lipopolysaccharide (LPS) stimulation experiments were performed in PBMCs of a non-septic kidney transplantation cohort (e:kid; n=10) obtained before and after organ-transplantation. Results : SOT patients demonstrated improved 30-day survival compared with non-SOT patients (84% vs. 66%; adjusted HR 0.49, 95% CI 0.26-0.95). Cytokine analysis revealed reduced IL-6 and TNF-α levels, alongside increased IL-18, indicating attenuation of hyperinflammation with preserved innate activation. Flow cytometry showed reductions in adaptive immune cells but relatively preserved innate immune function, reflected by higher monocytic HLA-DR expression. Proteomics identified enrichment of innate immune, complement, lipid metabolic response networks. At cellular level, SOT patients exhibited increased GLUT1 expression and enhanced TFAM/TFB2M interaction, indicating coordinated immunometabolism. Conclusion : Septic SOT patients display a reprogrammed immune response characterized by attenuation of hyperinflammation, preservation of innate immune competence, and coordinated immunometabolism. This immune pattern may underlie the observed survival advantage and provides a mechanistic framework for targeted therapeutic strategies in sepsis. Take Home Message: Septic patients with prior solid organ transplantation show improved 30-day survival despite chronic immunosuppression. This study provides the first comprehensive multilayer characterization of their immune response, demonstrating a reprogrammed profile with attenuated systemic inflammation, relative preservation of innate immune competence, and coordinated immunometabolism. These findings challenge the concept of global immunosuppression and suggest that distinct host-response patterns may contribute to outcome differences in sepsis.

(1) Background: Sepsis is characterized by profound heterogeneity of immune responses, complicating biomarker-based prediction of clinical outcomes. Latent human cytomegalovirus (HCMV) infection is one of the strongest modulators of the human immune system and may influence cytokine-mediated signaling during sepsis. (2) Methods: In this post hoc analysis of 331 patients from the prospective multicenter SepsisDataNet.NRW cohort (German Clinical Trial Registry No. DRKS00018871), we quantified 13 serum cytokines on day 1 after sepsis diagnosis and determined HCMV IgG serostatus via ELISA. Using nested cross-validated logistic regression with exhaustive feature selection, we identified cytokine panels predictive of 30-day survival in the total cohort and in subgroups stratified by HCMV serostatus. (3) Results: In the total cohort, a four-cytokine panel (IL-6, IL-10, TNF-α, IL-12p70) predicted 30-day survival with a cross-validated area under the curve (AUC) of 0.66 [95% CI: 0.59–0.72]. Stratification by HCMV serostatus revealed distinct predictive profiles: in HCMV-seropositive patients, a two-cytokine model (IL-10, IL-23) achieved an AUC of 0.69 [95% CI: 0.61–0.77], whereas in seronegative patients, a model based on IL-8 and IL-17A failed to generalize (AUC = 0.47 [95% CI: 0.33–0.61]). Kaplan–Meier analysis confirmed a significant separation of survival curves for the HCMV-seropositive group (p < 0.001) but not for seronegative patients (p = 0.282). (4) Conclusions: HCMV serostatus defines an immunological context in which cytokine-based prediction of sepsis outcome becomes feasible. These data suggest that viral serostatus should be systematically incorporated into biomarker discovery and immunophenotyping approaches to improve the reproducibility and biological interpretability of sepsis endotyping.

Sepsis is a life-threatening condition characterized by a dysregulated immune response to infection. Toll-like receptor 4 plays a central role in pathogen recognition and inflammatory signalling and has been considered a key driver of sepsis pathophysiology. Pharmacological inhibition of this receptor showed beneficial effects in experimental models but failed in clinical trials. We therefore aimed to quantify in vivo activation of Toll-like receptor 4 in patients with sepsis and to determine its association with 30-day survival. Peripheral blood mononuclear cells were obtained from 100 patients with sepsis enrolled in the SepsisDataNet.NRW cohort. Samples were collected on day 1 (within 36 h after diagnosis) and day 4. Activation of TLR4 was quantified by measuring receptor phosphorylation using a validated proximity ligation assay. Survival analyses were performed using Kaplan-Meier curves and Cox proportional hazards regression models to assess the association between receptor activation and 30-day mortality. Overall activation of TLR4 was low, with median values below one signal per cell at both day 1 and day 4. Despite the generally low levels, a subgroup of patients showed increased receptor activation. Higher activation was associated with significantly reduced 30-day survival. Patients with elevated activation had a higher risk of death both at day 1 (HR 2.03, 95

BackgroundSepsis is characterized by a dysregulated host response to infection, leading to concurrent hyperinflammation and immunosuppression, including profound alterations in T lymphocyte homeostasis. The chemokine CXCL10, an interferon-γ-inducible mediator of T cell trafficking, has been implicated in immune activation and tissue injury. However, it remains unclear whether genetic variation in CXCL10 contributes to T cell dysregulation and clinical outcomes in sepsis.MethodsIn a prospective cohort of septic patients (n=278), we analyzed CXCL10 rs8878 genotypes, circulating immune cell counts, cytokine concentrations, and CXCL10 protein and mRNA expression in whole blood. Associations between genotype, immune parameters, plasma proteomics and 30-day survival were assessed using group comparisons and Kaplan-Meier analyses. Correlation analyses were performed to evaluate relationships between CXCL10 concentrations, cytokines, and clinical parameters.ResultsVariants in the CXCL10 gene were associated with T cell dysregulation. Carriers of the rs8878 AA genotype exhibited higher circulating T cell counts and improved survival compared with G-allele carriers. Higher total and CD8+ T cell counts were significantly associated with improved survival. Among non-survivors, AA-genotype carriers showed increased CXCL10 mRNA expression, indicating genotype-dependent regulation of CXCL10 expression under conditions of fatal disease progression. CXCL10 concentrations on day 1 were positively correlated with multiple inflammatory cytokines, including IL-6, IL-8, IL-10, IL-18, MCP-1, IFN-γ, and interferon-α2, and inversely correlated with total T cell counts, supporting a link between CXCL10, systemic inflammation, and T cell depletion. No significant associations were observed between CXCL10 genotype and plasma proteomics and routine clinical parameters.ConclusionThe CXCL10 rs8878 genotype is associated with T cell dynamics and 30-day survival in sepsis, suggesting a genotype-dependent modulation of the adaptive immune response. While the AA genotype is linked to preserved T cell counts and improved outcomes, increased CXCL10 expression in non-survivors points to a context-dependent role in inflammation-driven immune dysregulation. These findings identify CXCL10 as a potential biomarker for risk stratification and a candidate target for immunomodulatory therapies in sepsis.

IntroductionRepair of mitochondrial damage seems pivotal for clinical recovery and determining outcome in patients with critical COVID-19. However, reliable biomarkers for non-invasively assessing mitochondrial repair in peripheral blood of critically ill COVID-19 patients are currently lacking. Accordingly, we sought to assess different surrogates of mitochondrial repair in peripheral blood and correlate these measurements with clinical outcome in patients with critical COVID-19.MethodsIn this prospective multicentric cohort study, 88 critically ill COVID-19 patients were enrolled across three German intensive care units. Gene products of mitochondrial quality control (MFN2, PINK, TFAM, TFB2M) and the mtDNA copy number were measured in peripheral blood mononuclear cells. Furthermore, the protein interactions between TFAM and TFB2M were quantified. Patients were stratified regarding 30-day mortality. ResultsTranscript levels of the assessed mRNA markers of mitochondrial quality control were not associated with clinical outcome. In contrast, more than 10.7 protein interactions per cell were associated with a 74% 30-day survival (37 out of 50), while 10.7 or fewer protein interactions per cell were associated with a 32% 30-day survival (12 out of 38; p < 0.001). Furthermore, multivariable Cox regression analysis revealed TFAM-TFB2M protein interaction as an independent predictor for 30-day survival (HR: 3.2; 95% CI: 1.6 to 6.5; p < 0.001). DiscussionOur findings indicate that TFAM-TFB2M protein interactions, identified as a novel biomarker, are strongly and independently associated with 30-day survival in critical COVID-19. Therefore, our data suggest a significant impact of mitochondrial repair and quality control on clinical outcome in critical COVID-19.

The glucocorticoid receptor (GR), particularly its isoforms GRα and GRβ, plays a crucial role in modulating inflammatory responses. The rs6198 single nucleotide polymorphism (SNP) in the NR3C1 gene, which encodes GR, has been associated with adverse outcomes in various diseases due to its potential effect on GR isoform expression. This study aims to explore the impact of the rs6198 SNP in sepsis. Specifically, we tested the hypothesis that the presence of a particular genotype of the rs6198 SNP is associated with an increased 30-day mortality rate in patients with sepsis. This prospective, multicenter study included 204 ICU patients diagnosed with sepsis, as part of the Sepsis.Data.Net NRW cohort. Genotyping for rs6198 and immunofluorescence as well as quantification of GR expression were performed. Statistical analyses included Hardy-Weinberg equilibrium, Kaplan-Meier survival analysis, log-rank tests, multivariate Cox regression, and logistic regression. Genotyping for the rs6198 SNP identified 137 patients (67%) with the TT- and 67 (33%) with CC/CT-genotype. Patients with the TT-genotype had a 30-day survival rate of 65% (89 of 137 patients), which was significantly lower than the 82% survival rate (55 of 67 patients) observed in the patients with the CC/CT-genotype (p = 0.006). A multivariate Cox regression analysis, adjusted for age, SOFA and SAPS2 score, and selected laboratory values, revealed that the TT-genotype was independently associated with an increased risk of death (HR 3.56, 95% CI 1.22-10.38, p = 0.02). Subgroup analysis demonstrated a particularly pronounced impact among patients with initially high disease severity (HR 6.16, 95% CI 1.66-22.80, p = 0.007). In addition, expression analysis revealed a significantly higher presence of GRα in patients with the TT-genotype compared to those with CC/CT genotype (p = 0.023). Increased GRα expression was also associated with higher 30-day mortality (HR 2.38, 95% CI 1.48-3.82, p < 0.001). The rs6198 SNP in the NR3C1 gene is associated with 30-day mortality in sepsis patients and correlates with increased expression of the GRα isoform. These results highlight the TT-genotype as a potential risk marker. Further research is needed to clarify the causal mechanisms and explore personalized therapeutic implications in sepsis management.

Sepsis is a common life-threatening disease caused by dysregulated immune response and metabolic acidosis which lead to organ failure. An abnormal expression of aquaporins plays an important role in organ failure. Additionally, genetic variants in aquaporins impact on the outcome in sepsis. Thus, we investigated the polymorphism (rs17553719) and expression of aquaporin-3 (AQP3) and correlated these measurements with the survival of sepsis patients. Accordingly, we collected blood samples on several days (plus clinical data) from 265 sepsis patients who stayed in different ICUs in Germany. Serum plasma, DNA, and RNA were then separated to detect the promotor genotypes of AQP3 mRNA expression of AQP3 and several cytokines. The results showed that the homozygote CC genotype exhibited a significant decrease in 30-day survival (38.9%) compared to the CT (66.15%) and TT genotypes (76.3%) (p = 0.003). Moreover, AQP3 mRNA expression was significantly higher and nearly doubled in the CC compared to the CT (p = 0.0044) and TT genotypes (p = 0.018) on the day of study inclusion. This was accompanied by an increased IL-33 concentration in the CC genotype (day 0: p = 0.0026 and day 3: p = 0.008). In summary, the C allele of the AQP3 polymorphism (rs17553719) shows an association with increased AQP3 expression and IL-33 concentration accompanied by decreased survival in patients with sepsis.

Background Metabolic derangements in sepsis stem from mitochondrial injury and contribute to organ dysfunction and mortality. Thus, repair of mitochondrial damage seems pivotal for recovery and determining clinical outcome in sepsis. However, reliable biomarkers assessing mitochondrial repair noninvasively in peripheral blood are currently lacking. Research Question Are different gene transcripts related to mitochondrial repair (ie, biogenesis, fusion, fission, mitophagy) and the protein interaction assessing mitochondrial biogenesis, both measured in peripheral blood, associated with disease severity and clinical outcome? Study Design and Methods Healthy control patients (n = 22), uninfected critically ill control patients (n = 13), and patients with sepsis (n = 75) were included in this prospective multicentric observational study. Gene products of mitochondrial quality control and mitochondrial DNA were measured on day 1 and 4 in peripheral blood mononuclear cells. In addition, we assessed in the same samples the mitochondrial protein interaction of mitochondrial transcription factor A (TFAM)-mitochondrial transcription factor B2 (TFB2M) using a proximity ligation assay. Patients with sepsis were stratified in the outcome-related subgroups ICU-free within 1 week (n = 16), not ICU-free within 1 week (n = 36), and 30-day nonsurvivors (n = 23). Results Transcript levels of the assessed messenger RNA markers of patients with sepsis were not associated with disease severity nor did they predict clinical outcome. Strikingly, the mitochondrial protein interaction of TFAM-TFB2M on day 4 (P < .05) and the difference between day 1 and 4 (P < .001) allowed stratification in the three clinical outcome subgroups. In addition, a decline in TFAM-TFB2M protein interactions between day 1 and 4 was an independent predicator for 30-day mortality (adjusted hazard ratio, 8.34; 95% CI, 2.73-25.45; P < .001). Interpretation Patients with sepsis with an early activation of mitochondrial biogenesis were more likely to be ICU-free within 1 week. A mitochondrial and clinical recovery can be assessed via the protein interaction of TFAM-TFB2M in peripheral blood. Thus, mitochondrial protein interactions targeting mitochondrial biogenesis provide a promising dimension of novel biomarkers assessing mitochondrial dysfunction in sepsis.

The variability in mortality in sepsis could be a consequence of genetic variability. The glucocorticoid system and the intermediate TSC22D3 gene product—glucocorticoid-induced leucine zipper—are clinically relevant in sepsis, which is why this study aimed to clarify whether TSC22D3 gene polymorphisms contribute to the variance in sepsis mortality. Blood samples for DNA extraction were obtained from 455 patients with a sepsis diagnosis according to the Sepsis-III criteria and from 73 control subjects. A SNP TaqMan assay was used to detect single-nucleotide polymorphisms (SNPs) in the TSC22D3 gene. Statistical and graphical analyses were performed using the SPSS Statistics and GraphPad Prism software. C-allele carriers of rs3747406 have a 2.07-fold higher mortality rate when the sequential organ failure assessment (SOFA) score is higher than eight. In a multivariate COX regression model, the SNP rs3747406 with a SOFA score ≥ 8 was found to be an independent risk factor for 30-day survival in sepsis. The HR was calculated to be 2.12, with a p-value of 0.011. The wild-type allele was present in four out of six SNPs in our cohort. The promoter of TSC22D3 was found to be highly conserved. However, we discovered that the C-allele of rs3747406 poses a risk for sepsis mortality for SOFA Scores higher than 6.

Sepsis involves an immunological systemic response to a microbial pathogenic insult, leading to a cascade of interconnected biochemical, cellular, and organ–organ interaction networks. Potential drug targets can depict aquaporins, as they are involved in immunological processes. In immune cells, AQP3 and AQP9 are of special interest. In this study, we tested the hypothesis that these aquaporins are expressed in the blood cells of septic patients and impact sepsis survival. Clinical data, routine laboratory parameters, and blood samples from septic patients were analyzed on day 1 and day 8 after sepsis diagnosis. AQP expression and cytokine serum concentrations were measured. AQP3 mRNA expression increased over the duration of sepsis and was correlated with lymphocyte count. High AQP3 expression was associated with increased survival. In contrast, AQP9 expression was not altered during sepsis and was correlated with neutrophil count, and low levels of AQP9 were associated with increased survival. Furthermore, AQP9 expression was an independent risk factor for sepsis lethality. In conclusion, AQP3 and AQP9 may play contrary roles in the pathophysiology of sepsis, and these results suggest that AQP9 may be a novel drug target in sepsis and, concurrently, a valuable biomarker of the disease.

Sepsis presents a challenge due to its complex immune responses, where balance between inflammation and anti-inflammation is critical for survival. Glucocorticoid-induced leucine zipper (GILZ) is key protein in achieving this balance, suppressing inflammation and mediating glucocorticoid response. This study aims to investigate GILZ transcript variants in sepsis patients and explore their potential for patient stratification and optimizing glucocorticoid therapy. Sepsis patients meeting the criteria outlined in Sepsis-3 were enrolled, and RNA was isolated from whole blood samples. Quantitative mRNA expression of GILZ transcript variants in both sepsis patient samples (n = 121) and the monocytic U937 cell line (n = 3), treated with hydrocortisone and lipopolysaccharides, was assessed using quantitative PCR (qPCR). Elevated expression of GILZ transcript variant 1 (GILZ TV 1) serves as a marker for heightened 30-day mortality in septic patients. Increased levels of GILZ TV 1 within the initial day of sepsis onset are associated with a 2.2-[95

Sepsis is a life-threatening condition caused by the dysregulated host response to infection. Novel therapeutic options are urgently needed and aquaporin inhibitors could suffice as aquaporin 5 (Aqp5) knockdown provided enhanced sepsis survival in a murine sepsis model. Potential AQP5 inhibitors provide sulfonamides and their derivatives. In this study, we tested the hypothesis that sulfonamides reduce AQP5 expression in different conditions. The impact of sulfonamides on AQP5 expression and immune cell migration was examined in cell lines REH and RAW 264.7 by qPCR, Western blot and migration assay. Subsequently, whether furosemide and methazolamide are capable of reducing AQP5 expression after LPS incubation was investigated in whole blood samples of healthy volunteers. Incubation with methazolamide (10−5 M) and furosemide (10−6 M) reduced AQP5 mRNA and protein expression by about 30% in REH cells. Pre-incubation of the cells with methazolamide reduced cell migration towards SDF1-α compared to non-preincubated cells to control level. Pre-incubation with methazolamide in PBMCs led to a reduction in LPS-induced AQP5 expression compared to control levels, while furosemide failed to reduce it. Methazolamide appears to reduce AQP5 expression and migration of immune cells. However, after LPS administration, the reduction in AQP5 expression by methazolamide is no longer possible. Hence, our study indicates that methazolamide is capable of reducing AQP5 expression and has the potential to be used in sepsis prophylaxis.

Clinical success of Toll-Like receptor-4 (TLR-4) antagonists in sepsis therapy has thus far been lacking. As inhibition of a receptor can only be useful if the receptor is active, stratification of patients with active TLR-4 would be desirable. Our aim was to establish an assay to quantify phosphorylated TLR-4 using the proximity ligation assay (PLA). HEK293 TLR4/MD2/CD14 as well as THP-1 cells were stimulated with LPS and the activation of TLR-4 was measured using the PLA. Furthermore, peripheral blood mononuclear cells (PBMCs) from 25 sepsis patients were used to show the feasibility of this assay in clinical material. Activation of TLR-4 in these samples was compared to the PBMCs of 11 healthy individuals. We could show a transient activation of TLR-4 in both cell lines. Five min after the LPS stimulation, the signal increased 6.7-fold in the HEK293 cells and 4.3-fold in the THP-1 cells. The assay also worked well in the PBMCs of septic patients. Phosphorylation of TLR-4 at study inclusion was 2.9 times higher in septic patients compared to healthy volunteers. To conclude, we established a diagnostic assay that is able to quantify the phosphorylation of TLR-4 in cell culture and in clinical samples of sepsis patients. This makes large-scale stratification of sepsis patients for their TLR-4 activation status possible.

Midazolam is a widely used short-acting benzodiazepine. However, midazolam is also criticized for its deliriogenic potential. Since delirium is associated with a malfunction of the neurotransmitter acetylcholine, midazolam appears to interfere with its proper metabolism, which can be triggered by epigenetic modifications. Consequently, we tested the hypothesis that midazolam indeed changes the expression and activity of cholinergic genes by acetylcholinesterase assay and qPCR. Furthermore, we investigated the occurrence of changes in the epigenetic landscape by methylation specific PCR, ChiP-Assay and histone ELISA. In an in-vitro model containing SH-SY5Y neuroblastoma cells, U343 glioblastoma cells, and human peripheral blood mononuclear cells, we found that midazolam altered the activity of acetylcholinesterase /buturylcholinesterase (AChE / BChE). Interestingly, the increased expression of the buturylcholinesterase evoked by midazolam was accompanied by a reduced methylation of the BCHE gene and the di-methylation of histone 3 lysine 4 and came along with an increased expression of the lysine specific demethylase KDM1A. Last, inflammatory cytokines were not induced by midazolam. In conclusion, we found a promising mechanistic link between midazolam treatment and delirium, due to a significant disruption in cholinesterase homeostasis. In addition, midazolam seems to provoke profound changes in the epigenetic landscape. Therefore, our results can contribute to a better understanding of the hitherto poorly understood interactions and risk factors of midazolam on delirium.

The functionally important NF-κB1 promoter polymorphism (−94ins/delATTG) significantly shapes inflammation and impacts the outcome of sepsis. However, exploratory studies elucidating the molecular link of this genotype-dependent pattern are lacking. Accordingly, we analyzed lipopolysaccharide-stimulated peripheral blood mononuclear cells from both healthy volunteers (n = 20) and septic patients (n = 10). All individuals were genotyped for the −94ins/delATTG NF-κB1 promoter polymorphism. We found a diminished nuclear activity of the NF-κB subunit p50 in ID/DD genotypes after 48 h of lipopolysaccharide stimulation compared to II genotypes (p = 0.025). This was associated with higher TNF-α (p = 0.005) and interleukin 6 concentrations (p = 0.014) and an increased production of mitochondrial radical oxygen species in ID/DD genotypes (p = 0.001). Although ID/DD genotypes showed enhanced activation of mitochondrial biogenesis, they still had a significantly diminished cellular ATP content (p = 0.046) and lower mtDNA copy numbers (p = 0.010) compared to II genotypes. Strikingly, these findings were mirrored in peripheral blood mononuclear cells taken from septic patients. Our results emphasize the crucial aspect of considering NF-κB subunits in sepsis. We showed here that the deletion allele of the NF-κB1 (−94ins/delATTG) polymorphism was associated with the lower nuclear activity of subunit p50, which, in turn, was associated with aggravated inflammation and mitochondrial dysfunction.

The quantity of aquaporin 5 protein in neutrophil granulocytes is associated with human sepsis-survival. The C-allele of the aquaporin (AQP5)-1364A/C polymorphism was shown to be associated with decreased AQP5 expression, which was shown to be relevant in this context leading towards improved outcomes in sepsis. To date, the underlying mechanism of the C-allele-leading to lower AQP5 expression-has been unknown. Knowing the detailed mechanism depicts a crucial step with a target to further interventions. Genotype-dependent regulation of AQP5 expression might be mediated by the epigenetic mechanism of promoter methylation and treatment with epigenetic-drugs could maybe provide benefit. Hence, we tested the hypothesis that AQP5 promoter methylation differs between genotypes in specific types of immune cells.: AQP5 promoter methylation was quantified in cells of septic patients and controls by methylation-specific polymerase chain reaction and quantified by a standard curve. In cell-line models, AQP5 expression was analyzed after demethylation to determine the impact of promoter methylation on AQP5 expression. C-allele of AQP5-1364 A/C promoter polymorphism is associated with a five-fold increased promoter methylation in neutrophils (p = 0.0055) and a four-fold increase in monocytes (p = 0.0005) and lymphocytes (p = 0.0184) in septic patients and healthy controls as well. In addition, a decreased AQP5 promoter methylation was accompanied by an increased AQP5 expression in HL-60 (p = 0.0102) and REH cells (p = 0.0102). The C-allele which is associated with lower gene expression in sepsis is accompanied by a higher methylation level of the AQP5 promoter. Hence, AQP5 promoter methylation could depict a key mechanism in genotype-dependent expression.

Sepsis is characterized by a dysregulated immune response, metabolic derangements and bioenergetic failure. These alterations are closely associated with a profound and persisting mitochondrial dysfunction. This however occurs despite increased expression of the nuclear-encoded transcription factor A (TFAM) that normally supports mitochondrial biogenesis and functional recovery. Since this paradox may relate to an altered intracellular distribution of TFAM in sepsis, we tested the hypothesis that enhanced extramitochondrial TFAM expression does not translate into increased intramitochondrial TFAM abundance. Accordingly, we prospectively analyzed PBMCs both from septic patients (n = 10) and lipopolysaccharide stimulated PBMCs from healthy volunteers (n = 20). Extramitochondrial TFAM protein expression in sepsis patients was 1.8-fold greater compared to controls (p = 0.001), whereas intramitochondrial TFAM abundance was approximate 80% less (p < 0.001). This was accompanied by lower mitochondrial DNA copy numbers (p < 0.001), mtND1 expression (p < 0.001) and cellular ATP content (p < 0.001) in sepsis patients. These findings were mirrored in lipopolysaccharide stimulated PBMCs taken from healthy volunteers. Furthermore, TFAM-TFB2M protein interaction within the human mitochondrial core transcription initiation complex, was 74% lower in septic patients (p < 0.001). In conclusion, our findings, which demonstrate a diminished mitochondrial TFAM abundance in sepsis and endotoxemia, may help to explain the paradox of lacking bioenergetic recovery despite enhanced TFAM expression.

Mitochondrial DNA (mtDNA) plays a vital role as a damage-associated molecular pattern in sepsis being able to shape the immune response. Since pathogen recognition receptors of innate immune cells are activated by demethylated DNA only, we set out to investigate the amount of DNA methyltransferase 1 (DNMT1) in mitochondria and the extent of mtDNA methylation in a human endotoxin model. Peripheral blood mononuclear cells of 20 healthy individuals were isolated from whole blood and stimulated with lipopolysaccharide (LPS) for 48 h. Subsequently, DNMT1 protein abundance was assessed in whole cells and a mitochondrial fraction. At the same time, methylation levels of mtDNA were quantified, and cytokine expression in the supernatant was measured. Despite increased cellular expression of DNMT1 after LPS stimulation, the degree of mtDNA methylation slightly decreased. Strikingly the mitochondrial protein abundance of DNMT1 was reduced by 50% in line with the lower degree of mtDNA methylation. Although only modest alterations were seen in the degree of mtDNA methylation, these strongly correlated with IL-6 and IL-10 expression. Our data may hint at a protein import problem for DNMT1 into the mitochondria under LPS stimulation and suggest a role of demethylated mtDNA in the regulation of the inflammatory immune response.